Eugen Gheorghiu

Magneto-plasmonic biosensor with enhanced analytical response and stability.

We present novel solutions to surpass current analytic limitations of Magneto-Optical Surface Plasmon Resonance (MOSPR) assays, concerning both the chip structure and the method for data analysis. The structure of the chip is modified to contain a thin layer of Co-Au alloy instead of successive layers of homogeneous metals, as currently used. This alloy presents improved plasmonic and magnetic properties, yet a structural stability similar to Au-SPR chips, allowing for bioaffinity assays in saline solutions. Analyzing the whole reflectivity curve at multiple angles of incidence instead of the reflectivity value at a single incidence angle provides a high signal-to-noise ratio suitable for detection of minute analyte concentrations. Based on assessment of solutions with known refractive indices as well as of a model biomolecular interaction (i.e. IgG-AntiIgG) we demonstrate that the proposed structure of the MOSPR sensing chip and the procedure of data analysis allows for long-time assessment in liquid media with increased sensitivity over standard SPR analyses.

Quantitative assessment of specific carbonic anhydrase inhibitors effect on hypoxic cells using electrical impedance assays

Abstract Carbonic anhydrase IX (CA IX) is an important orchestrator of hypoxic tumour environment, associated with tumour progression, high incidence of metastasis and poor response to therapy. Due to its tumour specificity and involvement in associated pathological processes: tumourigenesis, angiogenesis, inhibiting CA IX enzymatic activity has become a valid therapeutic option. Dynamic cell-based biosensing platforms can complement cell-free and end-point analyses and supports the process of design and selection of potent and selective inhibitors. In this context, we assess the effectiveness of recently emerged CA IX inhibitors (sulphonamides and sulphocoumarins) and their antitumour potential using an electrical impedance spectroscopy biosensing platform. The analysis allows discriminating between the inhibitory capacities of the compounds and their inhibition mechanisms. Microscopy and biochemical assays complemented the analysis and validated impedance findings establishing a powerful biosensing tool for the evaluation of carbonic anhydrase inhibitors potency, effective for the screening and design of anticancer pharmacological agents.

Biodynsensing: Sensing Through Dynamics of Hybrid Affinity/Cellular Platforms; Towards Appraisal of Environmental and Biological Risks of Nanobiotechnology

Chemical cues and nano-topographies present on the surface or in the extracellular medium strongly influence the fate and adhesion of biological cells. Careful tuning of cell-matrix interaction via engineered surfaces, either attractive or repulsive, require non-invasive, long time monitoring capabilities and lay the foundation of sensing platforms for risk assessment. Aiming to assess changes underwent by biointerfaces due to cell-environment interaction (in particular nanotechnology products), we have developed hybrid cellular platforms allowing for time based dual assays, i.e., impedance/dielectric spectroscopy (IS) and Surface Plasmon Resonance (SPR). Such platforms comprising Flow Injection Analysis (FIA) have been advanced to assess the interaction between selected (normal and malignant) cells and nano-patterned and/or chemically modified surfaces, as well as the impact of engineered nanoparticles, revealed by the related changes exhibited by cell membrane, morphology, adhesion and monolayer integrity. Besides experimental aspects dealing with measurement set-up, we will emphasize theoretical aspects related to: dielectric modeling. Aiming for a quantitative approach, microscopic models on dielectric behavior of ensembles of interconnected cells have been developed and their capabilities will be outlined within the presentation. Assessment of affinity reactions as revealed by dielectric/impedance assays of biointerfaces. Modeling the dynamics of the impedance in relation to the quality of cell layer and sensors active surface, this study presents further developments of our approach described in Analytical Chemistry, 2002. Data analysis. This issue is related to the following basic question: Are there simple Biosensing Platforms? When coping with cellular platforms, either in suspension or immobilized (on filters, adhered on surfaces or entrapped, e.g., on using set-ups) there is an intrinsic nonlinear behavior of biological systems related to cellular mechanisms involved in sensing, i.e., adaptation to stimuli. This should not mean that when coping with living cells, stray effects might not also corrupt the measurement itself, introducing distinct dynamics. Besides targeted/

Multi Frequency, Multi Channel, Differential Impedance Analyzer for Rapid Assays

We have developed a multi-channel, multi-sine, differential Impedance Spectrometer as a low cost, fast instrument to monitor the impedance of electrode/specimen interface, at low frequencies (1 – 200 Hz), for assessment of the fast events (down to few seconds/measurement) involved in biosensing. Emphasis will be given to presenting the command and control software of the Multi-Sine Impedance Spectrometer, developed in LabVIEW, with Fourier Transform Core and options for application and analysis of multi-sine and/or single sine waveforms. Using Fourier Transform algorithm and Inverse Fourier Transform, a whole spectrum (1-200 Hz) can be recorded in seconds. The command and control software of the Multi-Sine Impedance Spectrometer enables as well the successive application and analysis of up to 8 sinusoidal waves with different frequencies rendering compatibility with previously developed set-ups and measurement protocols for biosensor assessment.

Biosensing Based on Magneto-Optical Surface Plasmon Resonance

In spite of the high analytic potential of Magneto Optical Surface Plasmon Resonance (MOSPR) assays, their applicability to biosensing has been limited due to significant chip stability issues. We present novel solutions to surpass current limitations of MOSPR sensing assays, based on innovative chip structure, tailored measurements and improved data analysis methods. The structure of the chip is modified to contain a thin layer of Co-Au alloy instead of successive layers of homogenous metals with magnetic and plasmonic properties, as currently used. This new approach presents improved plasmonic and magnetic properties, yet a structural stability similar to standard Au-SPR chips, allowing for bioaffinity assays in saline solutions. Moreover, using a custom-designed measurement configuration that allows the acquisition of the SPR curve, i.e., the reflectivity measured at multiple angles of incidence, instead of the reflectivity value at a single-incidence angle, a high signal-to-noise ratio is achieved, suitable for detection of minute analyte concentrations. The proposed structure of the MOSPR sensing chip and the procedure of data analysis allow for long time assessment in liquid media, a significant advancement over existing MOSPR chips, and confirm the MOSPR increased sensitivity over standard SPR analyses.

Sensing the cell- substrate interaction towards development of "smart" surfaces

Chemical cues and nanotopographies present on the surface strongly influence the fate and adhesion of biological cells. Careful tuning of the engineered surfaces, either attractant or repulsive, require non-invasive, long time monitoring capabilities.

High speed CMOS acquisition system based on FPGA embedded image processing for electro-optical measurements

Electro-optical measurements, i.e., optical waveguides and plasmonic based electrochemical impedance spectroscopy (P-EIS), are based on the sensitive dependence of refractive index of electro-optical sensors on surface charge density, modulated by an AC electrical field applied to the sensor surface. Recently, P-EIS has emerged as a new analytical tool that can resolve local impedance with high, optical spatial resolution, without using microelectrodes. This study describes a high speed image acquisition and processing system for electro-optical measurements, based on a high speed complementary metal-oxide semiconductor (CMOS) sensor and a field-programmable gate array (FPGA) board. The FPGA is used to configure CMOS parameters, as well as to receive and locally process the acquired images by performing Fourier analysis for each pixel, deriving the real and imaginary parts of the Fourier coefficients for the AC field frequencies. An AC field generator, for single or multi-sine signals, is synchronized with the high speed acquisition system for phase measurements. The system was successfully used for real-time angle-resolved electro-plasmonic measurements from 30 Hz up to 10 kHz, providing results consistent to ones obtained by a conventional electrical impedance approach. The system was able to detect amplitude variations with a relative variation of ±1%, even for rather low sampling rates per period (i.e., 8 samples per period). The PC (personal computer) acquisition and control software allows synchronized acquisition for multiple FPGA boards, making it also suitable for simultaneous angle-resolved P-EIS imaging.

High Performance Low Cost Impedance Spectrometer for Biosensing

Cell culture assays have become a routine testing method in the medical and pharmaceutical research. Real-time monitoring of cells on a substrate using electrical impedance is a convenient and non-invasive method of analysis. Cell proliferation and apoptosis can be quantified and characterized by electrical impedance in a time-dependent manner. Unfortunately monitoring several cell cultures at the same time is challenging because of the prohibitive costs of impedance analysis equipment or rather slow response if multiplexing the front-end of an impedance analyzer. We propose a low cost high performance impedance analyzer based on a two channel delta-sigma 16-bit ADC. The device can handle frequencies between 30 Hz and 10 kHz with input impedances ranging: 100mOhms - 10Mohms. The Automatic Level Control (ALC) algorithm allows for steady non-invasive voltage levels on the measured impedance independent of measuring frequency and impedance value. The device prototype performance is compared to commercial analyzers used for monitoring cell growth and related cell based e.g. biosensing applications. Low production cost and robustness to component tolerances owed to special digital processing inside the Digital Signal Controller (DSC) make this device very suitable to mass production, the first step in becoming a readily available tool.

Dynamic assessment of Amyloid oligomers – cell membrane interaction by advanced impedance spectroscopy

The amyloid β (Aβ) peptides are believed to be pivotal in Alzheimers disease (AD) pathogenesis and onset of vascular dysfunction. Recent studies indicate that Aβ1-42 treatment influences the expression of tight junction protein complexes, stress fibre formation, disruption and aggregation of actin filaments and cellular gap formation. Aiming for functional characterization of model cells upon Aβ1-42 treatment, we deployed an advanced Electric Cell-substrate Impedance Sensing for monitoring cell evolution. A precision Impedance Analyzer with a multiplexing module developed in house was used for recording individual electrode sets in the 40 Hz – 100 KHz frequency range. In a step forward from the classical ECIS assays, we report on a novel data analysis algorithm that enables access to cellular and paracellular electrical parameters and cell surface interaction with fully developed cell monolayers. The evolution of the impedance at selected frequencies provides evidence for a dual effect of Aβ42 exposure, at both paracellular permeability and cell adherence level, with intricate dynamics that open up new perspectives on Aβ1-42 oligomers – cell membrane interaction. Validation of electrical impedance assays of the amyloid fibrils effect on cell membrane structure is achieved by both AFM analysis and Surface Plasmon Resonance studies. The capabilities of this noninvasive, real time platform for cell analysis in a wider applicative context are outlined.

Revealing membrane potential by advanced impedance spectroscopy: theoretical and experimental aspects

In spite of recent advancement of novel optical and electrical techniques, availability of non-invasive, label-free methods to assess membrane potential of living cells is still an open issue. The theory linking membrane potential to the low frequency ? dispersion exhibited by suspensions of spherical shelled particles (presenting a net charge distribution on the inner side of the shell) has been pioneered in our previous studies with emphasis on the permittivity spectra. We now report on both theoretical and experimental aspects showing that whereas ? dispersion is related to a rather large variation exhibited by the permittivity spectrum the decrement presented by impedance magnitude spectrum is either extremely small, or occurs (for large cells) at very low frequencies (~mHz) explaining the lack of experimental bioimpedance data on the matter. Based on the microscopic model we indicate that an appropriate design of the experiment may enable access to membrane potential as well as to other relevant parameters when investigating living cells and charged lipid vesicles. We discuss the effect on the low frequency of permittivity and impedance spectra of: I. Parameters pertaining to cell membrane i.e. (i) membrane potential, (ii) size of the cells/vesicles, (iii) conductivity; II. Conductivity of the outer medium. A novel measuring set-up has recently been developed within the International Centre of Biodynamics allowing for sensitive low frequency (~10mHz) four point (bio)impedance assays. Its capability to test theoretical predictions is reported as well. The far reaching implications of this study applicability for life sciences (noninvasive access to the dynamics of relevant cell parameters) as well as for biosensing applications, e.g. assess the cytotoxicity of a wide range of stimuli, will be outlined.